Weng Y. Lee

Suppression of Reaginic Antibodies with Modified Allergens

Administration of multiple injections of conjugates of ovalbumin (OA) and polyethylene glycol (PEG) or its monomethoxy derivative (mPEG) into mice which had been sensitized with 2,4-dinitrophenylated OA (DNP3–OA) abrogated both the anti-OA and anti-DNP IgE responses, in spite of additional injections of the sensitizing dose of DNP3-OA in the presence of Al(OH)3. Treatment of mice with OA-PEG in Al(OH)3 stimulated preferentially helper T cells, whereas injection of mice with OA-PEG in the absence of adjuvant elicited predominantly suppressor T cells. The unresponsive state of mice which had been treated 21 days earlier with OA-PEG could not be broken by the transfer of normal spleen cells and an additional sensitizing dose of DNP3-OA. Transfer of spleen cells from tolerized animals to normal mice dampened the capacity of the latter to mount both anti-DNP and anti-OA IgE responses; however, the suppressive effect of these cells was eliminated by treatment of the normal recipients with cyclophosphamide, which is a procedure known to inactive suppressor T cells, and hence it may be concluded that this effect was not due to the carryover of the tolerogen with the transferred cells. All these results provide strong support for the conclusion that the suppressor cells induced by the treatment of mice with OA-PEG and OA-mPEG conjugates belonged to a T cell subpopulation, and that the B cells of these mice were devoid of suppressive activity.

Abrogation of the antibenzylpenicilloyl (BPO) IgE response with BPO-polyvinyl alcohol conjugates.

IgE antibodies to the benzylpenicilloyl (BPO) determinant and ovalbumin (OA) were readily induced B6D2F1 mice by a single i.p. injection of 10 µg of BPO4-OA suspended with 1 mg AI(OH)3 in 0.5 ml of saline. The potential of these mice to mount a de novo anti-BPO IgE antibody response was specifically abrogated by pretreatment of mice with tolerogenic conjugates consisting of the hapten coupled to the hydrophilic and nonimmunogenic polymers, polyvinyl alcohols (PVA), with average molecular weigths of 10,000 or 14,000 daltons. The average epitope density of these conjugates varied from 1.1 to 3.9. It is noteworthy that even BPO1.1-PVA, which elicied only weak PCA reactions in rats sensitized i.d. with mouse anti-BPO reaginic antibodies, was highly tolerogenic. More importantly, the ongoing anti-BPO reaginic response in sensitized mice was also readily suppressed by administration of these tolerogenic conjugates, e.g. a single dose of 100 µg of BPO2.7-PVA was sufficient to abolish an ongoing anti-BPO IgE response. Moreover, administration of these tolerogenic conjugates into mice sensitized to BPO4-OA, prior to their resensitization in presence of Bordetella pertussis, protected these animals from anaphylactic death on i.v. challenge with 2 mg of the polyvalent BPO9-MγG conjugate (MγG = mosue γ-globulins); by contrast, all control mice which had not received this tolerogenic regimen died of anaphylaxis. It is suggested that conjugates of PVA with haptenic antibiotic or other drug molecules may prove to be effective therapeutic agents for the specific suppression of IgE antibodies mediating allergies of the immediate type to a wide spectrum of low molecular weight allergenic compounds.

Suppression of the IgE antibody response to ovalbumin in mice with a conjugate of ovalbumin and isologous γ-globulins☆

Abstract Injection of a conjugate of ovalhumin (OA) with mouse γ-globulins (MγG) into normal B 6 D 2 F 1 mice, or into mice preimmunized with DNP 3 -OA and Al(OH) 3 , markedly depressed the capacity of these two groups of mice to mount, respectively, primary or secondary anti-DNP and anti-OA IgE antibody responses. The immunosuppressive effect of OA-Mγ G conjugates was specific for both the antigenic and carrier functions of OA, i.e., it did not affect the ability of the mice to mount anti-DNP and anti-ASC IgE responses upon immunization with DNP-ASC in Al(OH) 3 , Injection of unmodified OA or of conjugates of OA with rabbit γ-globulins, on the other hand, interfered only in a transient manner with the full expression of the primary anti-OA and anti-DNP IgE responses elicited by immunization with DNP 3 -OA and AI(OH) 3 . In experiments designed to elucidate the mechanism underlying this mode of immunosuppression, 3 × 10 7 spleen cells from mice, which had received four repeated ip injections of OA-MγG 4 weeks before sacrifice, were injected ip into normal nonirradiated syngeneic recipients, which received two injections of DNP 3 -OA and Al(OH) 3 4 hr and 4 weeks after cell transfer. The primary and secondary anti-DNP and anti-OA IgE responses of the recipients were profoundly suppressed by comparison with the responses of recipients of cells from normal or immunized mice. Moreover, transfer of spleen cells from normal or immune animals into mice which had been immunosuppressed with OA-MγG did not restore the ability of the recipients to mount anti-OA and anti-DNP reaginic responses. These results suggest, therefore, that the immunosuppression is at least in part due to suppressor cells which are induced by conjugates of isologous γ-globulins with a protein antigen.

Suppression of IgE antibody production in sensitized mice and rats by tolerogenic conjugates of synthetic hydrophilic polymers with antigen or hapten: effect on antigen-induced histamine release from peritoneal mast cells.

IgE antibodies were produced in mice and rats by immunization with ragweed pollen extract (RAG) or dinitrophenylated ovalbumin (DNP3-OA). Treatment of these animals with tolerogenic conjugates of (i) the antigen (RAG or OA) with monomethoxypolyethylene glycol (mPEG), or (ii) DNP with polyvinyl alcohol (DNP-PVA) resulted, within 7-14 days, in a fall in circulating IgE antibodies and in mast cell sensitivity, as assessed by the radioallergosorbent test (RAST) and in the in vitro antigen-induced histamine release (HR) test, respectively. The reduction in responsiveness was more marked in mice than rats; 10 days after a booster immunization, the IgE antibody titres in the RAG-mPEG-treated group of mice were approximately 10-fold lower than in the saline-treated group, with a 100-fold difference in cell sensitivity. DNP-PVA treatment of mice produced a more than 10-fold reduction in IgE anti-DNP titres with a substantial reduction in histamine release.

Studies on rabbit ribonucleases.

Abstract Rabbit spleen and pancreatic RN-ses were purified from the crude tissue extracts by the sequential application of column chromatography on Sephadex G-75, DEAE-Sephadex A-50, SE-Sephadex C-50, and CM-Sephadex C-50. A 50- and 23-fold purification of the respective enzymes, with a percentage recovery of 54 and 60, was achieved. On the basis of the results of analytical ultracentrifugation and electrophoresis the purified RNases were judged homogeneous. These preparations were devoid of deoxyribonuclease, phosphatase, and phosphodiesterase activities. The optimum activity of both rabbit RNases was at pH 7.0, the Michaelis-Menten constants being 0.52 and 1.47 mg/ml for the spleen and pancreatic RNase, respectively. The enzymatic activity of these RNases was inhibited by the cations, Ag + , Cu 2+ , and Zn 2+ , but was not affected by Na + , K + , Ca 2+ , and Mg 2+ , by p -chloromercuribenzoate, or by chelating agents such as sodium citrate and EDTA. These RNases were shown to be basic proteins in terms of their amino acid compositions and their isoelectric points. The molecular weights of rabbit spleen and pancreatic RNases, estimated from their amino acid compositions as 14,305 and 13,971, respectively, were in accord with the values of 14,420 and 14,190 determined by gel filtration on Sephadex G-75.

Tolerization of Bϵ cells by conjugates of haptens and isologous γ-globulins

Abstract The anti-hapten IgE response induced in B6D2F1 mice by an ip injection of 1 μg of dinitrophenylated ovalbumin (DNP3-OA) with 1 mg A1(OH)3 was abrogated by an iv injection of 1 mg of a conjugate consisting of the hapten coupled to isologous γ-globulins (DNP8-MγG); the latter treatment did not affect the anti-OA IgE response induced by DNP3-OA. On the other hand, administration of deaggregated OA into mice resulted in incapacitating their potential to produce both anti-DNP and anti-OA antibodies in response to a sensitizing dose of DNP3-OA. Moreover, whereas the splenic cells of mice immunosuppressed by DNP8-MγG did not mount an anti-DNP IgE response on challenge with either DNP3-OA or the conjugate of DNP with constituents of Ascaris suum (DNP-ASC), the cells of mice tolerized with deaggregated OA were able to mount an anti-DNP IgE response on sensitization with DNP-ASC. In adoptive cell transfer experiments, it was demonstrated that (i) the anti-DNP IgE response was elicited with a mixture of thymus cells of tolerized mice and bone marrow cells of primed mice, but not by a mixture of thymus cells of primed mice and bone marrow cells of tolerized mice; (ii) the ability of tolerized spleen cells to produce anti-DNP IgE antibodies was restored when supplemented with bone marrow cells of primed mice, but not with thymus cells of primed mice; (iii) cooperation of splenic T cells of tolerized mice with splenic B cells of primed mice resulted in a marked anti-DNP IgE response, but the mixture of primed T cells and of tolerized B cells produced only a minimal response. From all these results, it is concluded that the immunosuppressive treatment resulting from the administration of DNP8-MγG was due to tolerization of Bϵ cells.